LNA oligonucleotide mediates an anti-inflammatory effect in autoimmune myocarditis via targeting lactate dehydrogenase B.

Treatment of myocarditis is often limited to symptomatic treatment due to unknown pathomechanisms. In order to identify new therapeutic approaches, the contribution of locked nucleic acid antisense oligonucleotides (LNA ASOs) in autoimmune myocarditis was investigated. Hence, A/J mice were immunized with cardiac troponin I (TnI) to induce experimental autoimmune myocarditis (EAM) and treated with LNA ASOs. The results showed an unexpected anti-inflammatory effect for one administered LNA ASO MB_1114 by reducing cardiac inflammation and fibrosis. The target sequence of MB_1114 was identified as lactate dehydrogenase B (mLDHB). For further analysis, mice received mLdhb-specific GapmeR during induction of EAM. Here, mice receiving the mLdhb-specific GapmeR showed increased protein levels of cardiac mLDHB and a reduced cardiac inflammation and fibrosis. The effect of increased cardiac mLDHB protein level was associated with a downregulation of genes of reactive oxygen species (ROS)-associated proteins, indicating a reduction in ROS. Here, the suppression of murine pro-apoptotic Bcl-2-associated X protein (mBax) was also observed. In our study, an unexpected anti-inflammatory effect of LNA ASO MB_1114 and mLdhb-specific GapmeR during induction of EAM could be demonstrated in vivo. This effect was associated with increased protein levels of cardiac mLDHB, mBax suppression and reduced ROS activation. Thus, LDHB and LNA ASOs may be considered as a promising target for directed therapy of myocarditis. Nevertheless, further investigations are necessary to clarify the mechanism of action of anti-inflammatory LDHB-triggered effects.

Heart-Specific Immune Responses in an Animal Model of Autoimmune-Related Myocarditis Mitigated by an Immunoproteasome Inhibitor and Genetic Ablation.

BACKGROUND: Immune checkpoint inhibitor (ICI) therapy is often accompanied by immune-related pathology, with an increasing occurrence of high-risk ICI-related myocarditis. Understanding the mechanisms involved in this side effect could enable the development of management strategies. In mouse models, immune checkpoints, such as PD-1 (programmed cell death protein 1), control the threshold of self-antigen responses directed against cardiac TnI (troponin I). We aimed to identify how the immunoproteasome, the main proteolytic machinery in immune cells harboring 3 distinct protease activities in the LMP2 (low-molecular-weight protein 2), LMP7 (low-molecular-weight protein 7), and MECL1 (multicatalytic endopeptidase complex subunit 1) subunit, affects TnI-directed autoimmune pathology of the heart. METHODS: TnI-directed autoimmune myocarditis (TnI-AM), a CD4+ T-cell-mediated disease, was induced in mice lacking all 3 immunoproteasome subunits (triple-ip-/-) or lacking either the gene encoding LMP2 and LMP7 by immunization with a cardiac TnI peptide. Alternatively, before induction of TnI-AM or after establishment of autoimmune myocarditis, mice were treated with the immunoproteasome inhibitor ONX 0914. Immune parameters defining heart-specific autoimmunity were investigated in experimental TnI-AM and in 2 cases of ICI-related myocarditis. RESULTS: All immunoproteasome-deficient strains showed mitigated autoimmune-related cardiac pathology with less inflammation, lower proinflammatory and chemotactic cytokines, less interleukin-17 production, and reduced fibrosis formation. Protection from TnI-directed autoimmune heart pathology with improved cardiac function in LMP7-/- mice involved a changed balance between effector and regulatory CD4+ T cells in the spleen, with CD4+ T cells from LMP7-/- mice showing a higher expression of inhibitory PD-1 molecules. Blocked immunoproteasome proteolysis, by treatment of TLR2 (Toll-like receptor 2)-engaged and TLR7 (Toll-like receptor 7)/TLR8 (Toll-like receptor 8)-engaged CD14+ monocytes with ONX 0914, diminished proinflammatory cytokine responses, thereby reducing the boost for the expansion of self-reactive CD4+ T cells. Correspondingly, in mice, ONX 0914 treatment reversed cardiac autoimmune pathology, preventing the induction and progression of TnI-AM when self-reactive CD4+ T cells were primed. The autoimmune signature during experimental TnI-AM, with high immunoproteasome expression, immunoglobulin G deposition, interleukin-17 production in heart tissue, and TnI-directed humoral autoimmune responses, was also present in 2 cases of ICI-related myocarditis, demonstrating the activation of heart-specific autoimmune reactions by ICI therapy. CONCLUSIONS: By reversing heart-specific autoimmune responses, immunoproteasome inhibitors applied to a mouse model demonstrate their potential to aid in the management of autoimmune myocarditis in humans, possibly including patients with ICI-related heart-specific autoimmunity.

FN14 Signaling Plays a Pathogenic Role in a Mouse Model of Experimental Autoimmune Myocarditis.

BACKGROUND: The pathogenesis of inflammatory cardiomyopathy is affected by the activation of autoimmune-mediated cascades. To study these cascades, we developed an experimental model of troponin I (TnI)-induced autoimmune myocarditis (EAM). One factor playing a pivotal role in the context of autoimmune disorders is the receptor fibroblast growth factor-inducible 14 (FN14). Thus, the impact of FN14 in the development of autoimmune myocarditis was investigated. METHODS AND RESULTS: TnI-immunization led to a significantly increased myocardial FN14 mRNA and protein expression in wild-type (wt) mice. To investigate the precise role of FN14 in EAM, FN14 knockout (ko) and wt littermates were immunized with TnI or control buffer. The animals were evaluated for cardiac parameters and indicators of myocardial injury. FN14 deficiency resulted in better cardiac performance, less myocardial inflammation, fibrosis, and cardiac damage. A lower myocardial mRNA expression of inflammatory cytokines and chemokines as well as their receptors could be demonstrated in TnI-immunized FN14ko compared to wt mice also immunized with TnI. Western blot analysis revealed a contribution of nuclear factor kappa-light-chain-enhancer of activated B cells to FN14-induced signaling cascades. CONCLUSIONS: In the pathogenesis of autoimmune myocarditis, the inflammatory response to cardiac injury is attenuated in FN14ko mice. Thus, inhibition of FN14 in patients might represent a novel therapeutic strategy in the treatment of inflammatory cardiomyopathy.

Adiponectin deficiency has no effect in murine autoimmune myocarditis.

BACKGROUND: Adiponectin is a hormone that together with its receptors modulates a number of metabolic processes including gluconeogenesis and lipid catabolism. It belongs to the C1QTNF (complement C1q tumor necrosis factor-related protein) family, which has a variety of members with high amino acid sequence homology and overlapping functions. Concentration of adiponectin in blood is inversely correlated with body fat percentage and cardiac risk factors like blood pressure and CRP (C-reactive protein) level. Studies have identified the existence of a cardiac adiponectin system. However, little is known about the role of this system in the pathogenesis of autoimmune myocarditis. Thus, we have studied the involvement of adiponectin in the development of this autoimmune disorder in a mouse model of experimental autoimmune myocarditis (EAM). METHODS: Adiponectin knockout (ko) and wild type (wt) mice were immunized with cardiac troponin I (cTnI) to induce an EAM. To determine the severity of myocardial damage, inflammation and fibrosis were scored after HE and Afog staining and high sensitivity troponin T (hsTnT) level was measured. To detect if changes in specific inflammatory cell numbers could be observed between the genotypes, we performed immunohistochemical staining to detect T lymphocytes, B lymphocytes and macrophages. The level of the humoral immune response was determined through the measurement of cTnI-specific serum IgG autoantibodies. Relative mRNA expression of different cytokines, C1QTNF family members and adiponectin receptors in the heart tissue was analyzed with qPCR. RESULTS: Animals immunized with cTnI developed autoimmune myocarditis with a significant deterioration of cardiac parameters compared to the corresponding control group. The adiponectin ko group immunized with cTnI showed a tendency towards increased inflammation, fibrosis, heart-to-body-weight ratio, infiltration pattern of T lymphocytes, B lymphocytes and macrophages, hsTnT concentration, humoral immune response and mRNA expression of interleukin 6 in the heart tissue and decreased weight gain compared to the wt group immunized with cTnI. However, the difference to the wt group treated with cTnI was not significant. The analysis of cardiac mRNA expression of adiponectin receptors and four C1QTNF family members, most suitable for fulfilling the functions of adiponectin in adiponectin ko mice, did not show any significant differences between adiponectin ko and wt group at all. CONCLUSION: Our study reveals that the absence of adiponectin did not lead to a significantly increased impairment of cardiac function and was also unlikely to be compensated by its receptors or other C1QTNF family members in the murine model of EAM. Here, other synergistic or redundant effects might play a role and must be investigated in further studies to understand the role and function of adiponectin in autoimmune myocarditis.

Critical role of RAGE and HMGB1 in inflammatory heart disease.

Autoimmune response to cardiac troponin I (TnI) induces inflammation and fibrosis in the myocardium. High-mobility group box 1 (HMGB1) is a multifunctional protein that exerts proinflammatory activity by mainly binding to receptor for advanced glycation end products (RAGE). The involvement of the HMGB1-RAGE axis in the pathogenesis of inflammatory cardiomyopathy is yet not fully understood. Using the well-established model of TnI-induced experimental autoimmune myocarditis (EAM), we demonstrated that both local and systemic HMGB1 protein expression was elevated in wild-type (wt) mice after TnI immunization. Additionally, pharmacological inhibition of HMGB1 using glycyrrhizin or anti-HMGB1 antibody reduced inflammation in hearts of TnI-immunized wt mice. Furthermore, RAGE knockout (RAGE-ko) mice immunized with TnI showed no structural or physiological signs of cardiac impairment. Moreover, cardiac overexpression of HMGB1 using adeno-associated virus (AAV) vectors induced inflammation in the hearts of both wt and RAGE-ko mice. Finally, patients with myocarditis displayed increased local and systemic HMGB1 and soluble RAGE (sRAGE) expression. Together, our study highlights that HMGB1 and its main receptor, RAGE, appear to be crucial factors in the pathogenesis of TnI-induced EAM, because inhibition of HMGB1 and ablation of RAGE suppressed inflammation in the heart. Moreover, the proinflammatory effect of HMGB1 is not necessarily dependent on RAGE only. Other receptors of HMGB1 such as Toll-like receptors (TLRs) may also be involved in disease pathogenesis. These findings could be confirmed by the clinical relevance of HMGB1 and sRAGE. Therefore, blockage of one of these molecules might represent a novel therapeutic strategy in the treatment of autoimmune myocarditis and inflammatory cardiomyopathy.

Identification of novel antigens contributing to autoimmunity in cardiovascular diseases.

In myocarditis and dilated cardiomyopathy (DCM) patients the immune system may play an important role in disease progression. In this study, we aimed to identify new antigens as a target for autoimmune response that might play a crucial role in these diseases. Therefore, a peptide-array was used to investigate antibody binding profiles in patients with autoimmune myocarditis or DCM compared to healthy controls and thus to identify disease relevant antigens. To analyze the pathogenicity of the identified antigens, an experimental autoimmune myocarditis (EAM) model was used. Hereby, 3 peptide sequences, derived from myosin-binding-protein-C (MYBPC) fast-type, RNA-binding-protein 20 (RBM20), and dystrophin, showed pathogenic effects on the myocardium of mice. In summary, 3 potentially cardiopathogenic peptides (MYBPC fast-type, RBM20, dystrophin) were identified. Thus, this study could serve as a basis for future investigations aimed at determining further antigens leading to pathogenic effects on the myocardium of DCM as well as myocarditis patients.

Mouse models of autoimmune diseases - autoimmune myocarditis.

Cardiovascular diseases are the leading cause of death in industrialized nations worldwide. Of all deaths resulting from cardiovascular diseases, 2% are caused by inflammatory heart disease; specifically, myocarditis. The etiology causing myocarditis still remains unclear. Both infectious and non-infectious factors are capable of triggering myocarditis. Acute myocarditis manifests itself in a variety of ways ranging from subclinical disease to sudden heart failure, as well as the occurrence of chest pain, palpitations, and syncope. Myocarditis can lead to dilated cardiomyopathy, this being the most frequent cause for heart transplantation. Since the underlying mechanism and the pathways behind the disease initiation and progression still need to be elucidated, the need for mouse models simulating the human disease is evident. Various mouse models are frequently used to study myocarditis. Inflammation of the myocardium as a result of infectious agents can be investigated with a widely used animal model where mice are infected with coxsackievirus B3. For autoimmune (non-viral) myocarditis, several mouse models (including induction with myosin or troponin I) have been established to better understand the role of autoantibodies and their influence on disease progression. With these different models, various phases of the disease can be investigated and these findings are used to develop more specific therapies that can be translated into the clinic as a "bench-to-bedside" approach.

The Novel Extracellular Cyclophilin A (CyPA) - Inhibitor MM284 Reduces Myocardial Inflammation and Remodeling in a Mouse Model of Troponin I -Induced Myocarditis.

Cyclophilins are a group of highly conserved cytosolic enzymes that have a peptidylprolyl cis/trans isomerase activity. Cyclophilin A (CyPA) can be secreted in the extracellular space by inflammatory cells and upon cell death. The presence of CyPA in patients with non-ischemic cardiomyopathy is associated with poor clinical prognosis. Here, we investigated the inhibition of extracellular CyPA in a mouse model of troponin I-induced autoimmune myocarditis using the strictly extracellular CyPA-inhibitor MM284. Since A/J mice develop severe inflammation and fibrosis after immunization with murine cardiac troponin I (mcTn I), we used this model to analyze the effects of an extracellular CyPA inhibition. As extracellular CyPA-inhibitor we used the recently described CsA-derivate MM284. In vitro studies confirmed that MM284 inhibits CyPA-induced monocytic migration and adhesion. A/J mice immunized with mcTnI were treated with MM284 or vehicle every second day. After 28 days, we found a considerable reduction of myocardial injury and fibrosis. Further analysis revealed a reduced myocardial presence of T-cells and macrophages compared to control treated animals. Whereas MMP-9 expression was reduced significantly by MM284, we observed no significant reduction of inflammatory cytokines such as IL-6 or TNFα. Extracellular CyPA plays an important role in autoimmune myocarditis for myocardial damage and fibrosis. Our data suggest a new pharmacological approach for the treatment of myocardial inflammation and reduction of cardiac fibrosis by inhibition of extracellular CyPA.

Successful use of mRNA-nucleofection for overexpression of interleukin-10 in murine monocytes/macrophages for anti-inflammatory therapy in a murine model of autoimmune myocarditis.

BACKGROUND: Overexpression of interleukin-10 (IL-10) in murine CD11b(+) monocytes/macrophages via GMP-adapted mRNA-nucleofection was expected to improve clinical outcome and reduce adverse side effects in autoimmune myocarditis. This study represents the proof of principle for a novel anti-inflammatory therapy using overexpression of IL-10 in murine monocytes/macrophages by mRNA-nucleofection for the treatment of autoimmune myocarditis. METHODS AND RESULTS: Autoimmune myocarditis was induced in A/J mice by subcutaneous immunization with troponin I. CD11b(+) monocytes/macrophages were isolated from the peritoneum and IL-10 was overexpressed by mRNA-nucleofection. These cells were injected intravenously. Myocardial inflammation was assessed via histological and immunohistochemical examinations. Myocardial fibrosis was analyzed with Masson's trichrome staining. Antitroponin I antibodies were determined within the serum. Physical performance was evaluated using a running wheel and echocardiography. In vitro overexpression of IL-10 in CD11b(+) monocytes/macrophages resulted in a 7-fold increased production of IL-10 (n=3). In vivo higher levels of IL-10 and less inflammation were detected within the myocardium of treated compared with control mice (n=4). IL-10-treated mice showed lower antitroponin I antibodies (n=10) and a better physical performance (n=10). CONCLUSIONS: Application of IL-10-overexpressing CD11b(+) monocytes/macrophages reduced inflammation and improved physical performance in a murine model of autoimmune myocarditis. Thus, the use of genetically modified monocytes/macrophages facilitated a targeted therapy of local inflammation and may reduce systemic side effects. Because the nucleofection technique is GMP adapted, an in vivo use in humans seems basically feasible and the transfer to other inflammatory diseases seems likely.